FAK downregulation suppresses stem-like properties and migration of human colorectal cancer cells

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase, which is overexpressed in colorectal cancer cells. FAK could be activated by phosphorylation to participate in the transduction of multiple signaling pathways and self-renewal of cancer stem cells. Whether the downregulation of FAK inhibits the metastasis in colorectal cancer through the weakening of stem cell-like properties and its mechanisms has yet to be established. CD44, CD133, c-Myc, Nanog, and OCT4 were known to mark colorectal cancer stem cell properties. In this study, AKT inhibitor (MK-2206 2HCl) or FAK inhibitor (PF-562271) decreased the expression of stem cell markers (Nanog, OCT4, CD133, CD44, c-Myc) and spheroid formation in colorectal cancer. Moreover, FAK and AKT protein was shown to interact verified by co-immunoprecipitation. Furthermore, downregulation of FAK, transfected Lenti-FAK-EGFP-miR to colorectal cancer cells, reduced p-AKT but not AKT and decreased the expression of stem cell markers and spheroid formation in colorectal cancer. In conclusion, we demonstrated that downregulation of FAK inhibited stem cell-like properties and migration of colorectal cancer cells partly due to altered modulation of AKT phosphorylation by FAK.


Introduction
During the initiation of metastasis, epithelial cancer cells undergo an epithelial-mesenchymal transition (EMT), and as a result become migratory and invasive mesenchymal-like cells while acquiring stem cell-like properties and therapy resistance [1,2]. Cancer stem cells are equipped with highly efficient DNA repair and effective redox tolerance systems in order to promote therapy resistance [3], and their number is increased outstandingly in cancers with cold immunity [4]. The prime time for medical intervention is the point when cancer stem cell-like cells start appearing. Early diagnosis of cancer-related symptoms is encouraged to understand and develop strategies for the treatment of colon cancer. It was known EMT is a feature of stem cell-like properties in tumor [2]. CD44, CD133, c-Myc, Nanog, OCT4 and SOX2 were known to mark colorectal cancer stem cell properties [5][6][7]. The transcription factor, Twist1, is a marker of the EMT which regulates matrix metalloproteinase (MMP) expression and has been identified as the downstream target of CD44 [8]. CD44 and CD133 were increased during the EMT of colorectal cancer cells [9]. C-Myc is the gene most strongly associated with cancer and positively regulates the expression of SnaiL, a master regulator of the EMT [10,11]. Furthermore, EMT facilitates the generation of cancer stem cell-like properties for metastasis, but also for self-renewal properties required for initiating secondary tumors attributed to Nanog, OCT4 and SOX [12].
Our previous study showed that Focal adhesion kinase (FAK) promotes the metastasis of colorectal cancer by inducing the EMT mediated by the AKT phosphorylation [13]. FAK is a cytoplasmic tyrosine kinase, which is overexpressed in cancer cells. FAK could be activated by phosphorylation to participate in the transduction of multiple signaling pathways and selfrenewal of cancer stem cells [14,15]. AKT was frequently dysregulated in tumors and played a pivotal role in tumor metastasis. Prevention of AKT phosphorylation is key to targeting cancer stem-like cells [16]. Recent studies have shown that the FAK/AKT pathway was activated in the process of obtaining stem cell-like properties of cancer cells [17,18]. Thus, we speculated that FAK may regulate AKT phosphorylation to promote the acquisition of stem cell-like properties and the migration of colorectal cancer.
The current study attempted to investigate that downregulation of FAK inhibits metastasis in colorectal cancer through the weakening of stem cell-like properties and its mechanisms. Expression of FAK or AKT was manipulated by transfection experiments and the use of specific inhibitors, and then the stem cell-like properties and migration of colorectal cancer cells were assessed.

Ethics statement
The research Ethics Committee of the Binzhou Medical University Hospital reviewed that the research content, scope and methods involved in this project meet the relevant requirements of medical ethics.

Western blotting analysis
Cells were collected by scraping and lysed with RIPA lysis buffer supplemented with 1% protease and 1% phosphatase inhibitor. Equal amounts of protein were separated by SDS-PAGE and blotted onto PVDF membranes. Membranes were scanned using an odyssey infrared imaging system (LI-COR, USA) and strips were analyzed with quantity one. Polyclonal antibodies against the following proteins were used: GAPDH (60004-1-1g,1:

Wound healing assay
To assess wound healing, cells were seeded into a 6-well cell culture plate and when 80%-90% confluency had been achieved, the cell populations were scratched by a 1 mm spearhead. Images were acquired after 0 h and 24 h of incubation and migration distance was measured by microscope software (Nikon, Japan).

Cell immunocytochemistry
Cells, at a density of 2x10 4 cells/ml, were plated onto slides fixed in a culture dish, followed by treatment with MK-2206 2HCl or PF-562271 or 0.9% NaCl for 24 h. Slides were fixed with icecold 100% methanol, quenched with 0.3% H 2 O 2 and blocked with normal goat serum. After incubation for 30 min with primary antibodies (as western blotting analysis) and washing, biotinylated secondary antibodies were added with incubation for 30 min, followed by washing and addition of preformed avidin/DH-biotinylated horseradish peroxidase H complex for 30 min. Slides were then overlain with DAB, rinsed, dried, mounted and a coverslip placed over them. Image pro plus analysis system was used to analyze protein expression. Three visual fields were randomly selected for each slide, and the total area and optical density were measured. Expression levels are expressed as average optical density (AOD): AOD = accumulated optical density/total area.

Co-Immunoprecipitation
Protein A/G agarose beads (78609, Pierce, USA) were washed with PBS, and diluted to 50% with PBS. A 100 μL aliquot of Protein G Sepharose (50%) and 1 μL non-immune serum were added to 1 mL cell lysate and the supernatant was collected after centrifugation. 10 μg rabbit anti-FAK antibody was added to the experimental groups and the same amount of rabbitderived IgG was to controls. 100 μL Protein G Sepharose (50%) was added to each sample and the agarose beads-antigen-antibody complex was collected. The complex was suspended in 100 μL 2×loading buffer and boiled for 5 min to release the antigen, antibody and Protein G beads. After centrifugation, the supernatant was subjected to electrophoresis and the whole protein lysate (Input), IgG eluted sample, and IP eluted sample were analyzed by western blotting. Finally, the protein signals were visualized using Pierce enhanced chemiluminescence reaction and exposed to medical X-ray film.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis
Extraction of total RNA and reverse transcription was performed using extraction (TIANGEN, China) and reverse transcription (TaKaRa, Japan), as previously reported [13]. FS universal SYBR green master (Roche, Germany) was used for cDNA synthesis via a fluorescence-based quantitative PCR method and real-time PCR System (ABI, USA). RT was performed using 2.0 μl 5X gDNA Eraser Buffer, 1.0 μl gDNA Eraser, 1 μg Total RNA and RNase Free dH2O to a total volume of 10 μl. The reaction conditions were 42˚C for 2 min and rapidly cooled to 4˚C. Subsequently, 10.0 μl reaction solution, 1.0 μl PrimeScript RT Enzyme Mix 1, 1.0 μl RT Primer Mix, 4.0 μl 5X PrimeScript Buffer 2 and 4.0 μl RNase Free dH2O were used. The reaction conditions were 37˚C for 15 min, 85˚C for 5 sec, rapidly cooled to 4˚C and stored at -20˚C. A fluorescence-based qPCR method was performed using 2 μl cDNA, 10 μl SYBR Green (Takara Bio, Inc.), 0.6 μl PCR forward primer (10 μM), 0.6 μl PCR reverse primer (10 μM) and 6.8 μl dH2O in a 20 μl PCR reaction volume. The RT-qPCR reaction was run on a StepOne Real--Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling parameters were as follows: Denaturing at 95˚C for 30 sec, 40 cycles at denaturing at 95˚C for 5 sec, primer annealing at 60˚C for 34 sec and extension temperature at 95˚C for 15 sec; final extension at 60˚C for 1 min and final denaturing at 95˚C for 15 sec. Gene expression levels were determined using the 2 -ΔΔCq method. Primers for GAPDH and FAK were from TaKaRa, Japan. GAPDH, Forward:  SPSS 20.0 software was used for all statistical analyses and PCR results were analyzed by the Mann-Whitney U test. Differences between experimental groups and controls were assessed using the Student's t-test and analysis of variance (ANOVA), followed by LSD's test. A value of p < 0.05 was considered significant.

Spheroid formation, cell migration and expression of FAK, p-FAK, AKT, p-AKT and stem cell marker proteins in HT29, HCT116, SW480, SW620 and RKO cells
Spheroid formation, cell migration and expression of FAK, p-FAK, AKT, p-AKT and stem cell markers in HT29, HCT116, SW480, SW620 and RKO cells were assessed to aid the selection of two suitable colorectal cancer cell-lines for subsequent experiments. The above five colorectal cancer cell lines had differing capacities for spheroid formation (Fig 1A). FAK, p-FAK, AKT, p-AKT, CD44, CD133, OCT4, Nanog and c-Myc proteins were expressed in all five cell-lines, although Sox2 was not. Expression of p-FAK and p-AKT was higher in HT29 and RKO than in HCT116, SW480 and SW620 cells (Fig 1B). In consideration of FAK or AKT being manipulated by transfection experiments and specificinhibitors in subsequent experiments, HT29 and RKO cells were adopted.

The effect of AKT inhibitor, MK-2206 2HCl, on survival of HT29 and RKO cells
On treatment with 25 uM MK-2206 2HCl for 24 h, viability of HT29 and RKO cells, measured by CCK8, was not significantly reduced (Fig 1C, p>0.05). At this time, MK-2206 2HCl could exert maximum efficacy in inhibiting target proteinexpression with minimal toxicity to thesurvival of HT29 or RKO cells.

MK-2206 2HCl inhibited stem-like properties and migration of colorectal cancer cells
Cell immunocytochemistry analysis showed that expression of p-AKT, CD44 and c-Myc proteins was decreased by 24 h treatment with 25 uM MK-2206 2HCl for RKO cells (Fig 2A,  p<0.05). In order to further clarify the impact of MK-2206 2HCl on the proteins expression of AKT, p-AKT and cancer stem cell markers, the proteins of HT29 and RKO cells were also detected by western blotting. Treatment with 25 uM MK-2206 2HCl for 24 h reduced expression of p-AKT, CD44, CD133, OCT4, Nanog and c-Myc (p<0.05) while that of AKT was unchanged (p>0.05) in HT29 and RKO cells (Fig 2B). In the meantime, the spheroid forming ability was reduced (Fig 2C, p<0.05), as was migration distance (Fig 2D, p<0.05), indicating loss of migratory capacity. It has been shown that p-AKT regulated the expression of cancer stem cell markers previously [16]. The above findings show that MK-2206 2HCl reduced stem

cell-like properties and migration of colorectal cancer cells via suppression of p-AKT expression.
The effect of FAK inhibitor, PF-562271, on survival of HT29 and RKO cells. Treatment with 5 uM PF-562271 for 24 h did not reduce HT29 and RKO cell viability as measured by CCK8 (Fig 1D, p>0.05). At this time, PF-562271 could exert maximum efficacy in inhibiting target protein expression with minimal toxicity to thesurvival of HT29 or RKO cells.

PF-562271 inhibited stem-like properties and migration of colorectal cancer cells though suppression of p-AKT expression
The RKO cells were treated with 5 uM PF-562271 for 24 h decreased p-FAK, p-AKT, CD44 and c-Myc expression by cell immunocytochemical analysis (Fig 3A, p<0.05) and expression of FAK, p-FAK, p-AKT, CD44, CD133, OCT4, Nanog and c-Myc by western blotting (p<0.05) in both HT29 and RKO cells while AKT was unchanged (p>0.05) (Fig 3B). In the meantime, HT29 and RKO spheroid formation was reduced (Fig 3C, p<0.05), as was migration distance (Fig 3D, p<0.05). The above findings supported that PF-562271 inhibited stem cell-like properties and migration of colorectal cancer cells perhaps via suppression of the FAK/p-FAK/p-AKT axis.

FAK regulated AKT phosphorylation in colorectal cancer cells
FAK and AKT proteins were shown to interact by co-immunoprecipitation in RKO cells ( Fig  4C). The RKO cells were treated with 5 uM PF-562271 for 24 h reduced p-AKT expression (p<0.05) but not that of AKT (p>0.05) (Fig 3B). Moreover, FAK knockdown in RKO cells reduced p-AKT expression (p<0.05) but not that of AKT (p>0.05) (Fig 4B). FAK appears to regulate AKT phosphorylation in colorectal cancer cells. In summary, the above findings show that the downregulation of FAK suppressed stem cell-like properties and migration of colorectal cancer cells partly by FAK modulation of AKT phosphorylation.

Discussion
Cancer stem cells represent a population of cells within a tumor with highly tumorigenic properties and may be identified by the expression of cancer stem cell markers. Cancer stem-like cells make a major contribution to metastasis and are known to exist in a variety of solid tumors, including colorectal cancer [19,20]. Targeting cancer stem-like cells appears to be a promising approach for the elimination of cancer recurrence and improvement of clinical outcomes [21].
AKT is thought to be involved in stem cell survival. Activation of AKT, such as the action of PI3K leading to phosphorylation of AKT to p-AKT, increased the number of neural stem cells [22,23]. The AKT/Nanog pathway is critical for the maintenance of sarcoma cancer stem cells and spheroid formation. Activation of the AKT/β-catenin pathway maintains the growth of cancer stem cells and directly modulates the expression of Nanog and OCT4 in colorectal cancer [24,25]. Inconsistently, some studies suggest that AKT is primarily induced in cancer stem cells through upregulation of the upstream Nanog [26]. A cross-regulation between AKT and Nanog may play a significant role in maintaining stem cell-like properties in tumors. In addition, AKT activation underpinned the stemness of colorectal cancer stem cells and CD133 expression changed when AKT was inhibited [23]. Increased CD44 expression in colorectal cancer cells was shown to be linked to increased AKT expression by the superimposition of targeted proteomic analysis on transcriptomic analysis [27,28]. C-Myc also was a downstream molecule in the AKT pathway and siRNA knockdown of c-Myc effectively inhibited cancer stem cell-like features [29]. In the current study, low expression of p-AKT was associated with decreased expression of stem cell markers, Nanog, OCT4, CD133, CD44 and c-Myc, in RKO and HT29 cells. Spheroid formation was suppressed by AKT inactivation. These data indicated colorectal cancer cells losed stem cell-like properties partly by inactivation of AKT with reduced phosphorylation of AKT.
FAK, which is implicated in tumor growth and metastasis, traffic to and is present in the nucleus, affecting gene regulation in a kinase-independent manner. The anti-tumor activity of FAK inhibitors has been used to reinforce traditional cytotoxic chemotherapy and immunotherapy in the form of combination treatments. PI3K activates AKT by phosphorylating serine 473 (Ser473) and the FAK/PI3K/AKT pathway is known to regulate cell motility. FAK was involved in the regulation of cell mobility via activation of the PI3K /AKT pathway, and associated phosphorylation of p85 subunits of tyrosine of PI3K in human cancer cells [28,30]. FAK and AKT proteins were shown to interact verified by co-immunoprecipitation and inhibition of FAK reduced p-AKT expression in this study. These data suggest a role for FAK in the regulation of AKT phosphorylation in colorectal cancer cells. FAK inhibition decreases cancer stem-like properties. FAK is implicated in modulating the resistant and aggressive phenotype of cancer stem cells [31]. The present study shows that FAK inhibitor decreased the expression of p-AKT and of stemness-related proteins, such as Nanog, OCT4, CD133, CD44 and c-Myc, in RKO and HT29 cells. When FAK expression of the transfected RKO cells was suppressed, levels of p-AKT and stemness markers were also decreased. FAK inhibition suppressed spheroid formation and migration of colorectal cancer cells.
In conclusion, the current study demonstrated that downregulation of FAK inhibited stem cell-like properties and migration of colorectal cancer cells partly due to altered modulation of AKT phosphorylation by FAK. Besides, pluripotency-associated factor Nanog expression was significantly attenuated by ERK knockdown [32]. FAK regulates cancer cell migration and stemness via ERK/Nanog pathway [33]. FAK is a potential tumor-suppressor target for colorectal cancer treatment, leading to reduced stem cells. Colorectal cancer stem-like cells are considered to be a primary cause of tumor recurrence. Elimination of cells with stem-like properties would make a significant contribution to improving clinical outcomes in patients with colorectal cancer.